Although, in vitro, cerebral endothelial cells can form tight junctions (TJ) and astrocytes can make and intercalate assemblies into their cell membrane, both structures are relatively few. By co-culturing these cells, we have found a structural interaction. When endothelial cells from beef brain, or their conditioned medium, are added to astrocyte cultures, some endothelial cells are joined by TJ that are about twice as extensive (average length of 5.4u) as are those in endothelial cells grown alone (average length of 2.8u). In co-cultures, 5 of the TJ measured were longer than 10u, the longest being 19.5u. The longest TJ in controls was no greater than 4.3u, to date. The TJ in co-cultures were also more complex: there were more strands and connections between them. Some astrocytes receiving medium conditioned by endothelial cells contained 5 to 10 times more asemblies than astrocytes maintained alone. Some of the assemblies were also clustered rather than randomly distributed. Thus, the addition of astrocytes results in the formation of TJ more nearly resembling in situ barrier junctions in their extent and complexity. Conversely, the addition of endothelium to astrocytes makes the number of assemblies more comparable to that of perivascular astrocytes in situ. However, the modulation of TJ and assemblies may be completely independent of one another.